Delivery system

ABSTRACT

Provided herein is a delivery system, including: (a) an optical sensor configured to detect data useful to create a map of a bodily surface; and (b) a printer operatively associated with the optical sensor and configured to deliver compositions (optionally including cells) to the bodily surface based upon the data or map. Methods of forming a tissue on a bodily surface of a patient in need thereof are also provided, as are methods, systems and computer program products useful for processing bodily surface data.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 61/293,481, filed Jan. 8, 2010,the disclosure of which is incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT SUPPORT

This work was supported by grant W81XWH-08-2-0032 from the Armed ForcesInstitute for Regenerative Medicine. The U.S. Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention concerns the in situ delivery of viable cells ontoa subject.

BACKGROUND OF THE INVENTION

In the United States, the mortality rate for burns is approximately 4.9%and increases dramatically with increasing total body surface area(TBSA) burned. The gold standard of treatment is the split-thicknessautograft, but this technique requires injuring one or more sites ofundamaged skin. Other treatment techniques include silver sulfadiazine,INTEGRA® (Johnson and Johnson, Hamburg, Germany; Integra Life SciencesCorporation, NJ), BIOBRANE® (Dow Hickam/Bertek Pharmaceuticals, SugarLand, Tex.), TRANSCYTE® (Advanced Tissue Sciences, Inc., La Jolla,Calif.), and allogeneic cells.

Large-scale manufacturing processes necessitate production of standardsizes of skin substitutes, but these standard-sized products cannotadequately cover irregular wounds. In addition, nearly all of thesetechniques require multiple surgical procedures, and are not ideal forlarge body surface area burns.

Allogeneic cell therapy can eliminate the need for autologous cellculture. Current delivery techniques include spraying cells onto thepatient or seeding a scaffold with cells before implantation. Cellspraying has been used to treat burns with autologous fibroblasts andkeratinocytes, but the delivery precision of current spraying technologyis low.

The ideal skin substitute possesses the following qualities: (1) itadheres intimately to the wound bed, especially for irregular surfaces;(2) it provides a non-antigenic microbial barrier; (3) it participatesin normal host repair mechanisms; (4) it maintains elasticity andlong-term durability; (5) it displays long-term mechanical and cosmeticfunction comparable to split-thickness autografts; (6) it requires asingle surgical procedure; (7) it is inexpensive; (8) it has anindefinite shelf life; and (9) it has minimal storage requirements.

New treatments are needed that better address the needs of burn woundpatients, as well as patient having other wounds and tissue injury ordisease.

SUMMARY OF THE INVENTION

Provided herein is a delivery system, including: (a) an optical sensorconfigured to detect data used to create a map of a bodily surface; and(b) a printer operatively associated with the optical sensor andconfigured to deliver cells and/or compositions to the bodily surfacebased upon the map. The sensor and printer can be associated with oneanother by connection of each to a common support or frame, to which mayalso be connected a subject support (e.g., a bed) to place a subject ina position for scanning of the subject's bodily surface. In someembodiments, the optical sensor includes a three-dimensional scanner. Insome embodiments, the optical sensor includes an infrared detector. Insome embodiments, the optical sensor is a laser scanner.

In some embodiments, the system further includes: (c) athree-dimensional plotter operatively connected with the optical sensor;and (d) a controller operatively connected with the printer.

In some embodiments, the printer includes a cartridge loaded with acomposition (e.g., a composition including cells, support compounds,growth factors, combinations thereof, etc.). In some embodiments, thecartridge includes a plurality of printheads, and wherein the cartridgeis in fluid communication with the plurality of printheads. In someembodiments, the printheads include nozzles configured forpressure-based delivery of cells and/or compositions.

Methods of forming a tissue on a bodily surface of a patient in needthereof are also provided, including: (a) scanning the bodily surface toobtain the three dimensional coordinates thereof; and then (b) printingviable cells on the bodily surface of the patient based upon thecoordinates to thereby form the tissue. In some embodiments, theprinting step is performed two or more times in sequence to make atissue having multiple layers.

Also provided are methods of processing bodily surface data obtainedfrom a three dimensional optical detector to provide a path to a printeroperatively associated to the optical detector, the methods including:interpreting the bodily surface data from the optical detector to form amodel of the bodily surface; transforming the model into a negative moldof the bodily surface, which mold is split into a plurality of Z-axislayers, which layers correspond to one or more tissue layers; andoverlaying each of the tissue layers with a series of lines which coverthe bodily surface, wherein the lines provide a path for the printer. Insome embodiments, the methods further include the step of obtaining thebodily surface data by scanning with a three-dimensional optical sensor.In some embodiments, the bodily surface data is wound surface data(e.g., skin wound surface data).

Further provided are systems for processing data of a bodily surfaceobtained from a three dimensional optical detector to provide a path toa printer operatively associated to the optical detector, the systemincluding: means for interpreting the bodily surface data from theoptical detector to form a model of the bodily surface; means fortransforming the model into a negative mold of the bodily surface, whichmold is split into a plurality of Z-axis layers, which layers correspondto one or more tissue layers; and means for overlaying each of thetissue layers with a series of lines which cover the bodily surface,wherein the lines provide a path for the printer. Some embodimentsfurther include means for obtaining the bodily surface data. In someembodiments, the bodily surface data is wound surface data (e.g., skinwound surface data).

Computer program products are also provided for processing data of abodily surface obtained from a three dimensional optical detector toprovide a path to a printer operatively associated to the opticaldetector, the computer program product including a computer readablemedium having computer readable program code embodied therein, thecomputer readable program code including: computer readable program codewhich interprets the bodily surface data from the optical detector toform a model of the bodily surface; computer readable program code whichtransforms the model into a negative mold of the bodily surface, whichmold is split into a plurality of Z-axis layers, which layers correspondto one or more tissue layers; and computer readable program code whichoverlays each of the tissue layers with a series of lines which coverthe bodily surface, wherein the lines provide a path for the printer. Insome embodiments, the bodily surface data is wound surface data (e.g.,skin wound surface data).

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate embodiments of the invention and,together with the description, serve to explain principles of theinvention.

FIG. 1. Perspective view of delivery system (5) having a printer support(10). This portable embodiment has wheels (24), and is positioned over asubject lying on a table (30) having a bed (35). The printer support isoperatively connected to members (23, 22, 21) configured to allow theprinter support (10) to be moveable about the Z axis (member 23), the Xaxis (member 22), and the Y axis (member 21).

FIG. 2. Bottom view of printer support (10) having optical sensor (11)and a plurality of printheads (12).

FIG. 3. Alternative embodiment of portable delivery system (5) having anattached computer (40), positioned over a subject lying on a table (30).

FIG. 4. Alternative embodiment of portable delivery system (5) having anattached light (50), positioned over a subject lying on a table (30)

FIG. 5. Alternative embodiment of portable delivery system (5) having anattached computer (40) and a cover (60) over the printer support (10),positioned over a subject lying on a table (30).

FIG. 6. Alternative embodiment of delivery system (5) having an attachedcomputer (40) and a cover (60) over the printer support (10). The system(5) is attached to a table (30).

FIG. 7. Cutout view of printer support (10) having a camera (11),infrared sensors (15), and a plurality of printer cartridges (14).

FIG. 8. Exploded view of a printer cartridge (14).

FIG. 9. Skin repair using bioprinting shows significant difference inwound size between 1 and 4 weeks after injury (p<0.05).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Provided herein and further described below are systems, compositions,devices and methods useful for the delivery of cells and tissues onto asubject in need thereof. In some embodiments, a cartridge based celldelivery system including a printer is provided, which printer isoperatively associated with a scanner.

The disclosures of all United States patent references cited herein arehereby incorporated by reference to the extent they are consistent withthe disclosure set forth herein. As used herein in the description ofthe invention and the appended claims, the singular forms “a,” “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. Furthermore, the terms “about” and“approximately” as used herein when referring to a measurable value suchas an amount of a compound, dose, time, temperature, and the like, ismeant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% ofthe specified amount. Also, as used herein, “and/or” or “/” refers toand encompasses any and all possible combinations of one or more of theassociated listed items, as well as the lack of combinations wheninterpreted in the alternative (“or”).

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the relevant art. It will be further understoodthat terms, such as those defined in commonly used dictionaries, shouldbe interpreted as having a meaning that is consistent with their meaningin the context of the specification and relevant art and should not beinterpreted in an idealized or overly formal sense unless expressly sodefined herein. Well-known functions or constructions may not bedescribed in detail for brevity and/or clarity.

The present invention is described herein, in part, with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems) and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer program instructions. These computer program instructions maybe provided to a processor of a general purpose computer, specialpurpose computer, or other programmable data processing apparatus toproduce a machine, such that the instructions, which execute via theprocessor of the computer or other programmable data processingapparatus, create means for implementing the functions/acts specified inthe flowchart and/or block diagram block or blocks.

As illustrated in FIG. 1, in some embodiments a delivery system (5) isprovided which includes a printer support (10) thereon. In someembodiments, the delivery system may be provided on wheels (24) forportability. The delivery system (5) may be positioned over a subjectlying on a table (30) having a bed (35) when in use. In someembodiments, a locking mechanism may be provided to lock the deliverysystem (5) in place relative to the table (30) or bed (35).

The printer support (10) according to some embodiments is operativelyconnected to members (23, 22, 21) configured to allow the printersupport (10) to be moveable about the Z axis (member 23), the X axis(member 22), and the Y axis (member 21).

As illustrated in FIG. 2, in some embodiments the printer support (10)includes an optical sensor (11) and a plurality of printheads (12). Inother embodiments, the printer support includes a plurality ofprintheads (12), but the optical sensor (11) is provided on a separatesupport (not shown).

Some alternative embodiments of the delivery system (5) are illustratedin FIGS. 3-6. FIG. 3 illustrates an embodiment of the delivery systemhaving an attached computer (40), positioned over a subject lying on atable (30). FIG. 4 illustrates another embodiment, and includes anattached light (50), positioned over a subject lying on a table (30).FIG. 5 illustrates an embodiment having an attached computer (40) and acover (60) over the printer support (10), positioned over a subjectlying on a table (30). FIG. 6 illustrates an embodiment of a deliverysystem (5) having an attached computer (40) and a cover (60) over theprinter support (10). The system (5) is attached to a table (30).

FIG. 7 illustrates an embodiment of a printer support (10) having acamera (11), infrared sensors (15), and a plurality of printercartridges (14).

An aspect of the present invention is a method of treating a wound(e.g., burns, abrasions, lacerations, incisions, pressure sores,puncture wounds, penetration wounds, gunshot wounds, crushing injuries,etc.) in a subject in need thereof, in which cells and/or compositionsare applied thereto in an amount effective to treat the wound.

Examples of wounds that can be treated with the present inventioninclude burn wounds. Burn wounds are tissue injuries that can resultfrom heat, chemicals, sunlight, electricity, radiation, etc. Burnscaused by heat, or thermal burns, are the most common. Chemical burnsresemble thermal burns. Though burn wounds tend to occur most often onthe skin, other body structures may be affected. For example, a severeburn may penetrate down to the fat, muscle or bone. In some embodiments,cells corresponding to one or more of these tissues may be deliveredonto the wound site, e.g., in a layer-by-layer application that mimicsthe natural tissues.

Wounds may be characterized by the depth of injury as known in the art.For example, the degree of a burn is characterized as first, second orthird depending on the depth of the tissues injured. In a first-degreeburn, only the top layer of skin (the epidermis) is damaged. Insecond-degree burns, the middle layer of skin (the dermis) is damaged.Finally, in a third-degree burn, the most severe type, the damage isdeep enough to affect the inner (fat) layer of the skin. Similarly,pressure sores of the skin are characterized as stage I (red, unbrokenskin, erythema does not fade with release of pressure), stage II(disrupted epidermis, often with invasion into the dermis), stage III(injury of the dermis), and stage IV (subcutaneous tissue is exposed).

In pressure sore wounds, pressure-induced constriction of localcapillaries results in ischemia in the affected skin. Similarly, a burnwound is ischemic due to associated capillary thrombosis. A diabeticulcer is another example of a poorly perfused wound. For these types ofwounds, where blood is not readily available to aid in the normal courseof wound healing, in some embodiments dead and/or injured tissue isremoved (debridement) prior to application of the cells as providedherein.

In some embodiments, compositions may include an antimicrobial agent todecrease the risk of infection. In some embodiments, compositions mayinclude analgesics or anesthetics for pain relief, surfactants,anti-inflammatory agents, etc. See, e.g., U.S. Pat. No. 6,562,326 toMiller. Methods of attenuating swelling, such as treatment with cold(e.g., cool water, ice, etc.) and elevation of the affected area, mayalso be used.

According to some embodiments, the device may be used for both openwounds and closed wounds. In the case of closed wounds, the scannerand/or delivery device may be allowed access to the wound site throughsurgical means, inclusive of endoscopic procedures.

In some embodiments, reapplication of the disclosed cells and/orcompositions may be performed as needed. Cleansing to remove bacteriaand debridement to remove necrotic debris may also be warranted duringthe course of treatment. Application of a moisturizing cream or ointmentmay be used to soften wound eschar in order to assist in debridement.

A. Printer.

In some embodiments, cells, proteins, support materials, combinationsthereof, etc., are delivered with a printer. “Printing” as used hereinrefers to the delivery of droplets of cells and/or compositions withsmall volumes, e.g., from 0.5 to 500 microLiters, or 5 to 100microLiters, or from 10 to 75 microLiters per droplet. In someembodiments, droplets have a volume ranging from 0.5 to 500 picoLiters,or 5 to 100 picoLiters, or from 10 to 75 picoLiters per droplet.Printing may be performed by, e.g., using standard printers with printheads that are modified as described herein. The “printhead” is thedevice in an inkjet printer that sprays droplets (e.g., ink, or as usedherein, cells and/or compositions).

In some embodiments, printing can provide a precise delivery of cells toa resolution of approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or 200 μm. Printing can also deliver specific cells to specifictarget sites using a layer-by-layer fabrication. Such layer-by-layerfabrication may in some embodiments be performed in situ on a subject inneed thereof, and involve multiple cell types arranged with precision.This is in contrast to cell seeding or spraying techniques, in whichcells are randomly applied over a large area.

A “cartridge” as used herein refers to a vessel or reservoir in whichcells and/or compositions may be held, and which is in fluidcommunication with one or more nozzles in one or more printheads. Thecartridge may include the reservoir and delivery mechanism in a singleunit, as in a traditional inkjet cartridge, or the reservoir anddelivery mechanism may be in separate units but connected such that theyare in fluid communication (e.g., through the use of tubing).

In some embodiments, one or more cell types and/or compositions areloaded into an individual cartridge. “Compositions” may include cells,carriers, support materials, macromolecules such as proteins, cytokines,growth factors, etc., or any combination thereof. Compositions may alsoinclude oxygen generating biomaterials. See PCT Publication WO2008/124126.

In some embodiments, each cartridge is configured to connect to multiplenozzles and/or printheads, in contrast to standard inkjet printing inwhich one printhead is connected to one cartridge. This allows arbitraryprinthead configurations that can conform to the needs of the treatment.It also increases the throughput of the system and provides a rapidmethod of sterilization by attaching a cartridge of cleaning fluid tothe printheads.

In some embodiments, the printheads contain pressure-based nozzles. Apressure-based delivery system according to some embodiments allows theprinter to remain a safe distance above the patient and to accommodate avariety of body types. As used herein, a “pressure-based” deliverysystem uses three components: the pressure source, material reservoirs,and delivery mechanism. In some embodiments, the delivery mechanism is aseries of voltage operated inkjet valves. The pressure source isoperatively connected to the reservoir, which is in fluid communicationwith the delivery mechanism. In some embodiments, a gas (e.g., air, airplus 5% CO₂, etc.) is pumped into empty space in the reservoir by thepressure source, which in turn drives the material in the reservoir(cells and/or compositions) into the delivery mechanism.

This pressure-based system may be preferable in some embodiments ascompared to inkjet cartridges in the context of in situ printing becauseit separates the reservoir and delivery mechanism. A traditional inkjetcartridge includes the reservoir and delivery mechanism in a singleunit. If either the reservoir or delivery mechanism fails, then theentire unit fails. Furthermore, inkjet cartridges must be filled andsealed prior to printing. If the inkjet cartridge is filled with cells,failure of the cartridge means the loss of all cells contained in thatcartridge. The pressure-based delivery system solves both of theseissues. The reservoir and delivery mechanism can be replacedindividually in the case of failure of either component. Material isonly pumped to the delivery mechanism when it is needed, so failure ofthe delivery mechanism does not result in the loss of all material inthe reservoir.

In addition, in some embodiments the pressure-based system can provide amethod of detecting and clearing clogged valves. By placing a pressuresensor at the end of the valve, the delivery pressure can be compared tothe applied pressure. If the difference between the two pressures islarger than a certain threshold, then the valve is clogged. Redirectingthe output of the valve to a waste reservoir and applying a large burstof pressure may be used to try and clear the valve. If the clearingprocess fails, then the system will detect this and can continueprinting without using that valve.

In some embodiments, the printer includes a two-dimensional (X-Y) orthree-dimensional (X-Y-Z) plotter (e.g., driven by step motors). In someembodiments, the print head is equipped with a DC solenoid inkjet valve.In some embodiments, one or more, or several, reservoirs for loadingcells is connected to the inkjet valve. In some embodiments, the cellsand/or compositions may be supplied from the reservoirs to the valve ornozzle by air pressure. In some embodiments, the print head may bemounted on an X-Y-Z plotter to allow precise deposition of cells onto ascaffold. Positioning of the XYZ plotter under the print head may becontrolled via a controller. In some embodiments, the controlleracquires the positioning information from software loaded on a computer.In some embodiments, the software converts the image of the target to afour-byte protocol, which is used to activate specific inkjet valves andcoordinate the X-Y-Z position.

Cells may be printed in the form of a composition that contains acarrier. The cells may be provided in the form of a suspension,solution, or any suitable form. The carrier may be a solid or a liquid,or both (e.g., a gel). In some embodiments, the cells are provided as asuspension in the carrier to reduce clumping of the cells. Supportcompounds, growth factors, etc., may be included in compositions havingcells and/or may be included in compositions without cells (but mayinclude a suitable carrier), as desired.

Suitable gels include, but are not limited to, agars, collagen, fibrin,hyaluronic acid, hydrogels, etc. Besides gels, other support compoundsmay also be utilized in the present invention. Extracellular matrixanalogs, for example, may be combined with support gels to optimize orfunctionalize the gel. One or more growth factors may also be included.In some embodiments a temperative sensitive gel may be used. Examples oftemperature sensitive gels include thermosensitive hydrogels andthermosensitive polymer gels (e.g., a poloxamer such as Pluronic® F-127(BASF corporation, Mont Olive, N.J.)). See also U.S. Pat. Nos.6,201,065, 6,482,435.

Examples of suitable liquid carriers include, but are not limited to,water, ionic buffer solutions (e.g., phosphate buffer solution, citratebuffer solution, etc.), liquid media (e.g., modified Eagle's medium(“MEM”), Hanks' Balanced Salts, etc.), and so forth. The use of a liquidor gel carrier in the cell composition may in some embodiments ensureadequate hydration and minimize evaporation of the cells after printing.

Cells may also be transfected (e.g., with a specific gene) with materialof interest. Useful genetic material may be, for example, geneticsequences that are capable of reducing or eliminating an immune responsein the host. For example, the expression of cell surface antigens suchas class I and class II histocompatibility antigens can be suppressed.This would allow the transplanted cells to have a reduced chance ofrejection by the host. Cells may also be transfected with a geneencoding one or more growth factors. According to some embodiments,cells may be transfected during the printing process. See PCTpublication WO 2008/153968 to Xu et al.

The present invention includes the printing of tissues by theappropriate combination of cell and support material, or two or three ormore different cell types typically found in a common tissue, preferablyalong with appropriate support compound or compounds, and optionallywith one or more appropriate growth factors. Cells, support compounds,and growth factors may be printed from separate nozzles or through thesame nozzle in a common composition, depending upon the particulartissue (or tissue substitute) being formed. Printing may besimultaneous, sequential, or any combination thereof. Some of theingredients may be printed in the form of a first pattern (e.g., anerodable or degradable support material), and some of the ingredientsmay be printed in the form of a second pattern (e.g., cells in a patterndifferent from the support, or two different cell types in a differentpattern). Again, the particular combination and manner of printing willdepend upon the particular tissue construct desired.

In alternative embodiments in which increased delivery precision isdesired, the printer uses thermal or piezoelectric printheads and/orinkjet cartridges for increased delivery precision. Methods andcompositions for the inkjet printing of viable cells are known anddescribed in, for example, U.S. Pat. No. 7,051,654 to Boland et al.;Wilson et al. (2003) The Anatomical Record Part A 272A: 491-496.

In some embodiments, cells/compositions are printed with a modifiedinkjet printer. Modifications may include, but are not limited to, meansto control the temperature, humidity, shear force, speed of printing,and firing frequency, by modifications of, e.g., the printer driversoftware and/or the physical makeup of the printer. See, e.g., Pardo etal. (2003) Langmuir 19:1462-1466; U.S. Pat. No. 7,051,654 to Boland etal. Not every modification suggested in these references will besuitable to a given application, as will be appreciated by those skilledin the art. For example, in some embodiments, printers are not modifiedby using new gear mount pillars with closer tolerances by adding ahorizontal support, changing the transistor in the circuit to one withhigher amplification, and reentering the horizontal position encoder.Also, in some embodiments, printer software is not modified to lower theresistive voltages to avoid heating of the solutions above 37° C.

In some embodiments, printers (e.g., the commercial printers HP695C andHP550C) may be modified as follows. The printer top cover may be removedand the sensor for the cover disabled. The paper feeding mechanism maybe disabled to allow printing of cells onto solid substrates (e.g.,scaffolds). The ink absorbing pads (which are on the right side of theHP695C and HP550C printers) may be removed (e.g., to avoid the padscontaminating the bottom of the print cartridges during the printingprocess). To offer the capability of the printer to print 3D constructs,a customized Z-axis module with a controlled elevator chamber may beadded.

In some embodiments, the printer is a thermal bubble inkjet printer. Ingeneral, in a thermal bubble inkjet printer, resistors create heat inthe print head, which vaporizes ink to create a bubble. As the bubbleexpands, some of the ink is pushed out of a nozzle onto the paper. Avacuum is created when the bubble collapses, which pulls more ink intothe print head from the cartridge. In the present invention, the ink isreplaced with, e.g., cells and/or compositions of interest (e.g., cellsin a liquid carrier), and the paper is replaced with a suitablesubstrate, e.g., an agar or collagen coated substrate, or a suitablescaffold. See, e.g., U.S. Pat. No. 6,537,567 to Niklasen et al.

In other embodiments, cells are printed using a piezoelectric crystalvibration print head. In general, a piezoelectric crystal receives anelectric charge that causes it to vibrate, forcing ink out of thenozzle, and pulling more ink into the reservoir. In the presentinvention, the ink is replaced with, e.g., cells and/or compositions ofinterest. Compared with the thermal inkjet printing, the piezo-basedinkjet printing usually requires more power and higher vibrationfrequencies. Typical commercial piezo-printers use frequencies up to 30kHz and power sources ranging from 12 to 100 Watts. Therefore, in someembodiments a piezoelectric crystal vibration print head is used, with avibrating frequency of 1, 5, 10 or 15, to 20, 25, 30, or 35 or more kHz,and power sources from 5, 10, 20, 50, 100, 120, or 150, to 200, 250,300, 350, or 375 or more Watts.

The cells may also be printed by other means, such as the methods andcompositions for forming three-dimensional structures by deposition ofviable cells described in U.S. Pat. No. 6,986,739 to Warren et al.

In some embodiments, the print head nozzles are each independentlybetween 0.05 and 200 μm in diameter, or between 0.5 and 100 μm indiameter, or between 10 and 70 μm, or between 20 and 60 μm in diameter.In further embodiments, the nozzles are each independently about 40 or50 μm in diameter. In still further embodiments, the nozzles are eachindependently between 0.1 or 0.5 and 2 or 3 mm. A plurality of nozzleswith the same or different diameters may be provided. A more narrownozzle may give greater precision delivery but low throughput, and viceversa. These may be provided according to cell type and/or precisiondesired. Though in some embodiments the nozzles have a circular opening,other suitable shapes may be used, e.g., oval, square, rectangle, etc.,without departing from the spirit of the invention.

In some embodiments, the cells/compositions are formulated to provide anencapsulated form upon printing. The encapsulation of cells in permeablecapsules is known, and described in, for example, U.S. Pat. No.6,783,964. For example, the cells may be encapsulated in a microcapsuleof from 50 or 100 μm to 1 or 2 mm in diameter that includes an internalcell-containing core of polysaccharide gum surrounded by a semipermeablemembrane; a microcapsule that includes alginate in combination withpolylysine, polyornithine, and combinations thereof. Other suitableencapsulating materials include, but are not limited to, those describedin U.S. Pat. No. 5,702,444.

“Encapsulated” cells are cells or small clusters of cells or tissue thatare surrounded by a selective membrane laminate that allows passage ofoxygen and other required metabolites, releases certain cell secretions(e.g., insulin), but limits the transport of the larger agents of thehost's immune system to prevent immune rejection. Encapsulation may beuseful for, e.g., the delivery of cells and/or tissues containingxenogeneic or allogeneic cells while reducing the risk of immunerejection in a host. This may be useful, e.g., to treat diseases due toinadequate or loss or secretory cell function, or ailments that wouldbenefit from the addition of certain secretory cells.“Microencapsulation” of cells is where one, two, three or several cellsare encapsulated. In some embodiments, each membrane encapsulates 10cells or less, preferably 5 cells or less, of at least 50, 70, 80, 90 or95% or more of the printed cells.

In some embodiments, two or more layers may be separately applied, withsubsequent layers applied to the top surface of previous layers. Thelayers can, in one embodiment, fuse or otherwise combine followingapplication or, alternatively, remain substantially separate and dividedfollowing application to the subject.

The thickness of a printed layer (e.g., cell layer, support layer, etc.)may generally vary depending on the desired application. For example, insome embodiments, the thickness of a layer containing cells is fromabout 2 micrometers to about 3 millimeters, and in some embodiments,from about 20 micrometers to about 100 micrometers. Further, asindicated above, support compounds, such as gels, may be used tofacilitate the survival of printed cells.

“Support compounds” which may be included in compositions may be anynaturally occurring or synthetic support compound, includingcombinations thereof, suitable for the particular tissue being printed.In general, the support compound is preferably physiologicallyacceptable or biocompatible. Suitable examples include, but are notlimited to, alginate, collagen (including collagen VI), elastin,keratin, fibronectin, proteoglycans, glycoproteins, polylactide,polyethylene glycol, polycaprolactone, polycolide, polydioxanone,polyacrylates, polysulfones, peptide sequences, proteins andderivatives, oligopeptides, gelatin, elastin, fibrin, laminin,polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates,polyanhydrides, polyamino acids carbohydrates, polysaccharides andmodified polysaccharides, and derivatives and copolymers thereof (see,e.g., U.S. Pat. Nos. 6,991,652 and 6,969,480) as well as inorganicmaterials such as glass such as bioactive glass, ceramic, silica,alumina, calcite, hydroxyapatite, calcium phosphate, bone, andcombinations of all of the foregoing.

When printing certain types of two-dimensional or three-dimensionaltissues or portions thereof, it is sometimes desired that any subsequentcell growth is substantially limited to a predefined region. Thus, toinhibit cell growth outside of this predefined region, compounds may beprinted or otherwise applied to the print area that inhibit cell growthand thus form a boundary for the printed pattern. Some examples ofsuitable compounds for this purpose include, but are not limited to,agarose, poly(isopropyl N-polyacrylamide) gels, and so forth.

In one embodiment, for instance, this “boundary technique” may beemployed to form a multi-layered, three-dimensional tube of cells, suchas blood vessels. For example, a cell suspension may be mixed with afirst gel (“Gel A”) in one nozzle, while a second gel (“Gel B”) isloaded into another nozzle. Gel A induces cell attachment and growth,while Gel B inhibits cell growth. To form a tube, Gel A and the cellsuspension are printed in a circular pattern with a diameter and widthcorresponding to the diameter and wall thickness of the tube, e.g., fromabout 3 to about 10 millimeters in diameter and from about 0.5 to about3 millimeters in wall thickness. The inner and outer patterns are linedby Gel B defining the borders of the cell growth. For example, a syringecontaining Gel A and “CHO” cells and a syringe containing Gel B may beconnected to the nozzle. Gel B is printed first and allowed to cool forabout 1 to 5 minutes. Gel A and CHO cells are then printed on theagarose substrate. This process may be repeated for each layer.

B. Optical Detector.

In some embodiments, the area or areas of interest onto which cellsand/or compositions are to be delivered is detected by an opticaldetector device to determine the two-dimensional and/orthree-dimensional map of the area of interest. The optical detector is,in some embodiments, operatively associated with an attached celldelivery device, such that the cell delivery pattern may be optimizedfor in situ delivery of the cells and/or compositions based upon suchmap.

“Map” as used herein refers to the two- and/or three-dimensional surfacemeasurements, coordinates, and/or any other data that may represent thetwo- and/or three-dimensional surface of an area of interest (e.g., awound). The map may be updated by scanning at any time, and/or in realtime during delivery of cells and/or compositions.

As used herein, the “optical detector” may comprise one or moredetectors that detect light at various wavelengths, e.g., visible light,infrared, ultraviolet, combinations thereof, etc. In some embodiments,both visible light and infrared light are detected.

In some embodiments, an optical detector such as a camera may be used tocapture images that coincide with the surface measurements of the areaof interest. In some embodiments, an image sensor is used to collectlight reflected from an object and generate an image of the object. Amirror and lens system may be combined with the imaging device to focusthe light reflected by the object onto the image sensor. The imagesensor may be one of a charge coupled device (CCD) and a complementarymetal oxide semiconductor (CMOS), typically arranged into an area array,although the invention is not so limited. The number of sensors, eachrepresenting a pixel (short for “picture element”), determine theresolution of the image taken. A pixel is the smallest unit that makesup a digital image, and can represent the shade and/or color of aportion of an image. The output of a set of image sensors may be encodedas a set of pixels to create a digital image. The digital image may bestored in a compressed format such as in a jpeg, tiff, and/or gifformat, among others. The image may then be stored in a digital storagedevice and may be displayed on a monitor by a display application.

In some embodiments, the optical detector is a three dimensional scannersuch as that described in U.S. Pat. No. 6,856,407 to Knighton et al.(incorporated by reference herein), in which depth data for athree-dimensional object may be calculated from an intensity differenceresulting from an intensity gradient projected on the object capturingan intensity at a location on a surface in a single pixel of an imagesensing array (ISA). The intensity may be converted into a measurementof distance to the location relative to a reference point independentlyof data from other pixels of the ISA and independent of time of flightof light reflected from the location to the single pixel. A plurality ofcaptures of the intensity at the location may be compared underdifferent conditions to compensate for non-homogeneous environments orsurfaces.

In some embodiments, the optical detector is a three dimensional scanneras described in U.S. Patent Application Publication No. 2005/0237581 toKnighton et al. (incorporated by reference herein). The scanning devicemay be used to generate three dimensional representation of an area ofinterest. As used herein, three dimensional representations may be anytype of digital modeling, abstraction and/or similar techniques that mayutilize depth maps, polygon meshes, parametric solids, point clouds andsimilar data structures to create and store a three dimensionalrepresentation of the scanned object. The scanner may include a lens orset of lenses to focus light on one or more image sensing arrays (ISA).In some embodiments, the ISAs may be a charged coupled device (CCD),complementary metal oxide semiconductor (CMOS) sensor, or similarimaging array. In some embodiments, lenses may be replaced by and/orsupplemented with a reflector, light guide and/or similar article. Byvarying the focal settings, different aspects of the relief of an objectmay be brought into focus on an ISA. In some embodiments, an opticalsystem having one or more optical elements distributes a same view of atarget to a plurality of ISA's, each having a different focal rangerelative to the target.

Stereovision may also be used. Traditional stereovision methods estimateshape by establishing spatial correspondence of pixels in a pair ofstereo images. A new concept called spacetime stereo has been developed,which extends the matching of stereo images into the time domain. Byusing both spatial and temporal appearance variations, it was shown thatmatching ambiguity could be reduced and accuracy could be increased. Theshortcoming of spacetime stereo or any other stereo vision method isthat matching of stereo images is time-consuming, therefore making itdifficult to reconstruct high-resolution 3D shapes from stereo images inreal time.

Further vision based surface mapping techniques may use structuredlight, which includes various coding methods and employs varying numberof coded patterns. Unlike stereo vision methods, structured lightmethods usually use processing algorithms that are much simpler.Therefore, it becomes possible to achieve real-time performance, i.e.,measurement and reconstruction. For real-time shape measurement, thereare basically two approaches. The first approach is to use a singlepattern, typically a color pattern. The use of this approach employs acolor-encoded Moire technique for high-speed 3D surface contourretrieval. Other techniques use a rainbow 3D camera for high-speed 3Dvision. Still others use a color structured light technique forhigh-speed scans of moving objects. Because these methods use color tocode the patterns, the shape measurement result is affected to varyingdegrees by the variations of the object surface color. In general,better accuracy is obtained by using more patterns.

Another structured light approach for real-time shape measurement is theuse of multiple coded patterns with rapid switching between them so thatthey could be captured in a short period of time. This approach has beenused and develops a real-time 3D model measurement system that uses fourpatterns coded with stripe boundary codes. Some embodiments may providean acquisition speed of about 15 fps, which is sufficient for scanningslowly moving objects. However, like any other binary-coding method, thespatial resolution of these methods is relatively low because the stripewidth must be larger than one pixel. Moreover, switching the patterns byrepeatedly loading patterns to the projector may limit the switchingspeed of the patterns and therefore the speed of shape measurement.

Another example of an optical detector is found in U.S. Pat. Nos.6,788,210 and 6,438,272 (incorporated by reference herein), whichprovide a vision system for real-time and high-speed 3D shapemeasurement, with full capability of providing fast updating of the 3Dsurface maps and maps of the curves indicating the treatment areas andpositioning markings such as tick marks of the said curves. In thismanner, the sensor may serve to close the present automated debridementsystem control loop and as the means to provide for safe operation ofthe system, by for example, shutting the treatment laser beam off whenthe error between the actual position and desired position of thetreatment laser beam is more than a selected (programmed) threshold.This method is based on a rapid phase-shifting technique. This techniqueuses three phase-shifted, sinusoidal grayscale fringe patterns toprovide pixel-level resolution. The patterns are projected to the objectwith a switching speed of 240 fps. This system takes full advantage ofthe single-chip DLP technology for rapid switching of three coded fringepatterns. A color fringe pattern with its red, green, and blue channelscoded with three different patterns is created by a PC. When thispattern is sent to a single-chip DLP projector, the projector projectsthe three color channels in sequence repeatedly and rapidly. Toeliminate the effect of color, color filters on the color wheel of theprojector are removed. As a result, the projected fringe patterns areall in grayscale. A properly synchronized high-speed B/W CCD camera isused to capture the images of each color channel from which 3Dinformation of the object surface is retrieved. A color CCD camera,which is synchronized with the projector and aligned with the B/Wcamera, is also used to take 2D color pictures of the object at a framerate of 26.7 fps for texture mapping. Together with the fast 3Dreconstruction algorithm and parallel processing software,high-resolution, real-time 3D shape measurement is realized at a framerate of up to 40 fps and a resolution of 532×500 points per frame. Othersystems for 3D shape measurement known in the art can also be used inthe system and methods of the present invention, such as thosecommercially available from Blue Hill Optical Technologies, located inNorwood, Mass. or Nutfield Technology, Windham, N.H.

For the projection of the computer-generated patterns, a single-chip DLPprojector is used, which produces images based on a digital lightswitching technique. With this system, a complex facial surface has beenmapped at 40 fps (the accuracy of the system being 0.1×0.1×0.1 mm),providing an excellent speed and resolution for the present automatedlaser debridement and treatment systems and the like.

The color image is produced by projecting the red, green, and bluechannels sequentially and repeatedly at a high speed. The three colorchannels are then integrated into a full color image. To take advantageof this projection mechanism of a single-chip DLP projector, a colorpattern which is a combination of three patterns in the red, green, andblue channels is created. The projector has no color filters for amonochrome mode of operation. As a result, when the color pattern issent to the projector, it is projected as three grayscale patterns,switching rapidly from channel to channel at 240 fps. A high-speed B/Wcamera, which is synchronized with the projector, is used to capture thethree patterns rapidly for real-time 3D shape measurement. An additionalcolor camera is used to capture images for texture mapping. To obtain 3Dmaps and color information simultaneously, multi-threading programmingis used to guarantee that two cameras work independently and that thetiming of image grabbing is only determined by the external triggersignal.

For more realistic rendering of the object surface, a color texturemapping method may be used that is based on a sinusoidal phase-shiftingmethod. In this method, the three fringe patterns have a phase shift of2π/3 between neighboring patterns. Since averaging the three fringepatterns washes out the fringes, a color image can be obtained withoutfringes by setting the exposure time of the color camera to oneprojection cycle or 12.5 ms.

These systems provide the capability of rapidly projecting and capturingthree coded patterns rapidly. The employed fast three-stepphase-shifting method provides a real-time 3D reconstruction speed andhigh measurement accuracy of the order of 0.1×0.1×0.1 mm. The sinusoidalphase-shifting method that has been used extensively in opticalmetrology to measure 3D shapes of objects at various scales. In thismethod, a series of phase-shifted sinusoidal fringe patterns arerecorded, from which the phase information at every pixel is obtained.This phase information helps determine the correspondence between theimage field and the projection field. Once this correspondence isdetermined, the 3D coordinate information of the object can be retrievedbased on triangulation. A number of different sinusoidal phase-shiftingalgorithms are available. A three-step phase-shifting algorithm similarto the traditional three-step algorithm may be used, which requiresthree phase-shifted images.

In some embodiments, the optical detector and printer are both mountedon a portable X-Y-Z plotting system.

C. Cells and Tissues.

Any type of cell may be printed using the methods herein, including, butnot limited to, mammalian cells (including mouse, rat, dog, cat, monkeyand human cells), including somatic cells, stem cells, progenitor cellsand differentiated cells, without limitation. Stem cells have theability to replicate through numerous population doublings (e.g., atleast 60-80), in some cases essentially indefinitely, and also have theability to differentiate into multiple cell types (e.g., is pluripotentor multipotent). It is also possible for cells to be transfected with acompound of interest that results in the cells becoming immortalized(i.e., able to double more than 50 times). For example, it has beenreported that mammalian cell transfection with telomerase reversetranscriptase (hTERT) can immortalize neural progenitor cells (See U.S.Pat. No. 7,150,989 to Goldman et al.).

“Embryonic stem cell” as used herein refers to a cell that is derivedfrom the inner cell mass of a blastocyst and that is pluripotent.

“Amniotic fluid stem cell” as used herein refers to a cell, or progenyof a cell, that (a) is found in, or is collected from, mammalianamniotic fluid, mammalian chorionic villus, and/or mammalian placentaltissue, or any other suitable tissue or fluid from a mammalian donor,(b) is pluripotent; (c) has substantial proliferative potential, (d)optionally, but preferably, does not require feeder cell layers to growin vitro, and/or (e) optionally, but preferably, specifically bindsc-kit antibodies (particularly at the time of collection, as the abilityof the cells to bind c-kit antibodies may be lost over time as the cellsare grown in vitro).

“Pluripotent” as used herein refers to a cell that has completedifferentiation versatility, e.g., the capacity to grow into any of theanimal's cell types. A pluripotent cell can be self-renewing, and canremain dormant or quiescent with a tissue. Unlike a totipotent cell(e.g., a fertilized, diploid egg cell) a pluripotent cell cannot usuallyform a new blastocyst.

“Multipotent” as used herein refers to a cell that has the capacity togrow into any of a subset of the corresponding animal cell types. Unlikea pluripotent cell, a multipotent cell does not have the capacity toform all of the cell types of the corresponding animal.

Cells may be autologous (i.e., from the very subject to which they willbe applied) syngeneic (i.e., genetically identical or closely related,so as to minimize tissue transplant rejection), allogeneic (i.e., from anon-genetically identical member of the same species) or xenogeneic(i.e., from a member of a different species). Syngeneic cells includethose that are autogeneic (i.e., from the subject to be treated) andisogenic (i.e., a genetically identical but different subject, e.g.,from an identical twin). Cells may be obtained from, e.g., a donor(either living or cadaveric) or derived from an established cell strainor cell line. For example, cells may be harvested from a donor usingstandard biopsy techniques known in the art.

According to some embodiments, at least a portion of the cells areviable after they are printed. “Viable cells” includes cells that adhereto a culture dish or other substrate and/or are capable of survival(e.g., proliferation). In some embodiments, at least 30, 40 or 50% ofthe total cells loaded are viable, and in further embodiments at least60, 70, 80, or 90% or more of the total cells loaded are viable afterprinting. Cell viability may be measured by any conventional means,e.g., the MTS assay, and at a reasonable time after printing, e.g., 1day after printing completion. Viability is measured upon incubationunder conditions known in the art to be optimal for survival of thecertain cells types present. For example, many eukaryotic cell types aretypically incubated in a suitable medium at 5% carbon dioxide (95%atmospheric air) and 37 degrees Celsius.

Various mechanisms may be employed to facilitate the survival of thecells during and/or after printing. Specifically, compounds may beutilized that support the printed cells by providing hydration,nutrients, and/or structural support. These compounds may be applied tothe substrate using conventional techniques, such as manually, in a washor bath, through vapor deposition (e.g., physical or chemical vapordeposition), etc. These compounds may also be combined with the cellsand/or compositions before and/or during printing, or may be printed orotherwise applied to the substrate (e.g., coated) as a separate layerbeneath, above, and/or between cell layers. For example, one suchsupport compound is a gel having a viscosity that is low enough underthe printing conditions to pass through the nozzle of the print head,and that can gel to a stable shape during and/or after printing. Suchviscosities are typically within the range of from about 0.5 to about 50centipoise, in some embodiments from about 1 to about 20 centipoise, andin some embodiments, from about 1 to about 10 centipoise. Some examplesof suitable gels that may be used in the present invention include, butare not limited to, agars, collagen, hydrogels, etc.

Another polymer used for hydrogels is alginate, a natural polysaccharideextracted from seaweed. One feature of alginate solutions is theirgelling properties in the presence of divalent cations (e.g., Mg++,Ca++, Sr++, Ba++).

Besides gels, other support compounds may also be utilized in thepresent invention. Extracellular matrix analogs, for example, may becombined with support gels to optimize or functionalize the gel. In someembodiments, one or more growth factors may also be introduced in theprinted arrays. For example, slow release microspheres that contain oneor more growth factors in various concentrations and sequences may becombined with the cells and/or composition. Other suitable supportcompounds might include those that aid in avoiding apoptosis andnecrosis of the developing structures. For example, survival factors(e.g., basic fibroblast growth factor) may be added. In addition,transient genetic modifications of cells having antiapoptotic (e.g.,bcl-2 and telomerase) and/or blocking pathways may be included incompositions printed. Adhesives may also be utilized to assist in thesurvival of the cells after printing. For instance, soft tissueadhesives, such a cyanoacrylate esters, fibrin sealant, and/orgelatin-resorcinol-formaldehyde glues, may be utilized to inhibitnascent constructs from being washed off or moved following the printingof a layer. In addition, adhesives, such as arginine-glycine-asparticacid (RGD) ligands, may enhance the adhesion of cells to a gellingpolymer or other support compound. Extracellular proteins, extracellularprotein analogs, etc., may also be utilized.

“Growth factor” may be any naturally occurring or synthetic growthfactor, including combinations thereof, suitable for the particulartissue or array being printed. Numerous growth factors are known.Examples include, but are not limited to, insulin-like growth factor(e.g., IGF-1), transforming growth factor-beta (TGF-beta),bone-morphogenetic protein, fibroblast growth factor, platelet derivedgrowth factor (PDGF), vascular endothelial growth factor (VEGF),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), epidermal growth factor, fibroblast growth factor (FGF) (numbers1, 2 and 3), osteopontin, bone morphogenetic protein-2, growth hormonessuch as somatotropin, cellular attractants and attachment agents, etc.,and mixtures thereof. See, e.g., U.S. Pat. Nos. 7,019,192; 6,995,013;and 6,923,833. For example, growth factor proteins may be provided inthe printed composition and/or encoded by plasmids transfected intoprinted cells.

In some embodiments, cells, compositions, support compounds, and/orgrowth factors may be printed from separate nozzles or through the samenozzle in a common composition, depending upon the particular tissue (ortissue substitute) being formed. Printing may be simultaneous,sequential, or any combination thereof. Some of the ingredients may beprinted in the form of a first pattern (e.g., an erodable or degradablesupport material), and some of the ingredients may be printed in theform of a second pattern (e.g., cells in a pattern different from thesupport, or two different cell types in a different pattern). Theparticular combination and manner of printing will depend upon theparticular tissue being printed.

In some embodiments, cells/compositions are printed onto a substrate,e.g., a biocompatible scaffold, which may be subsequently implanted intoor grafted onto a subject in need thereof. In other embodiments,cells/compositions of interest are directly printed in situ onto livingtissues in the body, with or without prior substrate application (e.g.,a layer of fibrin) in which the cells may attach.

In some embodiments, cells may be isolated from tissues of interest andcultured with techniques known in the art. “Isolated” as used hereinsignifies that the cells are placed into conditions other than theirnatural environment. Tissue or cells are “harvested” when initiallyisolated from a subject, e.g., a primary explant.

The “primary culture” is the first culture to become established afterseeding disaggregated cells or primary explants into a culture vessel.“Expanding” or “expansion” as used herein refers to an increase innumber of viable cells. Expanding may be accomplished by, e.g.,“growing” the cells through one or more cell cycles, wherein at least aportion of the cells divide to produce additional cells. “Growing” asused herein includes the culture of cells such that the cells remainviable, and may or may not include expansion and/or differentiation ofthe cells.

“Passaged in vitro” or “passaged” refers to the transfer or subcultureof a cell culture to a second culture vessel, usually implyingmechanical or enzymatic disaggregation, reseeding, and often divisioninto two or more daughter cultures, depending upon the rate ofproliferation. If the population is selected for a particular genotypeor phenotype, the culture becomes a “cell strain” upon subculture, i.e.,the culture is homogeneous and possesses desirable characteristics(e.g., the ability to express a certain protein or marker).

“Express” or “expression” of a protein or other biological marker meansthat a gene encoding the same of a precursor thereof is transcribed, andpreferably, translated. Typically, according to the present invention,expression of a coding region of a gene will result in production of theencoded polypeptide, such that the cell is “positive” for that proteinor other downstream biological marker.

“Skin cells” include those cells normally found in skin, and includeepidermal cells (e.g., keratinocytes, melanocytes, Merkel cells,Langerhan cells, etc., and any combination thereof) and dermal cells(e.g., fibroblasts, adipocytes, mast cells, macrophages, and anycombination thereof). Skin tissue may be formed to mimic natural skin bythe inclusion of melanocytes and dermal papilla cells. Skin tissueproduced by the process of the present invention is useful forimplantation into or on a subject to, for example, treat burns, andother wounds such as incisions, lacerations, and crush injuries (e.g.,postsurgical wounds, and posttraumatic wounds, venous leg ulcers,diabetic foot ulcers, etc.)

“Muscle cells” include those cells normally found in muscle tissue,including smooth muscle cells, cardiac muscle cells, skeletal musclecells (e.g., muscle fibers or myocytes, myoblasts, myotubes, etc.), andany combination thereof. Muscle cells/tissues produced by the processesdescribed herein are useful for, among other things, the treatment ofinjuries or defects affecting muscle tissue, and/or promote musclehealing.

“Cartilage cells” include those cells normally found in cartilage, whichcells include chondrocytes. “Chondrocytes” produce and maintain theextracellular matrix of cartilage, by, e.g., producing collagen andproteoglycans. Cartilage is a highly specialized connective tissue foundthroughout the body, and its primary function is to provide structuralsupport for surrounding tissues (e.g., in the ear and nose) or tocushion (e.g., in the trachea and articular joints). Types of cartilageinclude hyaline cartilage (articular joints, nose, trachea,intervertebral disks (NP), vertebral end plates), elastic cartilage(tendon insertion site, ligament insertion site, meniscus,intervertebral disks (AP)), costochondral cartilage (rib, growth plate),and fibrocartilage (ear). The loss of cartilage in a subject can beproblematic, as it has a very limited repair capacity. “Mesenchymal stemcells” or “MSCs” are progenitors of chondrocytes. MSCs can alsodifferentiate into osteoblasts. Cartilage cells/tissues produced by theprocesses described herein are useful for, among other things,implantation into a subject to treat cartilage injury or disease.

“Bone cells” include those cells normally found in bone, and includeosteoblasts, osteoclasts, osteocytes, and any combination thereof. Bonecells/tissues produced by the processes described herein are useful for,among other things, implantation into a subject to treat bone fracturesor defects, and/or promote bone healing.

“Nervous system cells” or “nerve cells” include those cells normallyfound in the peripheral nervous system, including neuronal and glialcells.

“Vascular cells” include those cells normally found in the mammalianvasculature, including blood vessels, and include endothelial cells,smooth muscle cells and fibroblasts.

In some embodiments, stem cells are printed onto substrates by inkjetprinting. Stein cells may be printed alone (typically in combinationwith a support compound or compounds) or in combination with one or moreadditional cells (e.g., in a combination selected to produce a tissue asdescribed above). In some embodiments, stem cells are differentiatedinto cells of interest.

“Differentiation” and “differentiating” as used herein include (a)treatment of the cells to induce differentiation and completion ofdifferentiation of the cells in response to such treatment, both priorto printing on a substrate, (b) treatment of the cells to inducedifferentiation, then printing of the cells on a substrate, and thendifferentiation of the cells in response to such treatment after theyhave been printed, (c) printing of the cells, simultaneously orsequentially, with a differentiation factor(s) that inducesdifferentiation after the cells have been printed, (d) contacting thecells after printing to differentiation factors or media, etc., andcombinations of all of the foregoing. In some embodiments,differentiation may be modulated or delayed by contacting an appropriatefactor or factors to the cell in like manner as described above. In someembodiments appropriate differentiation factors are one or more of thegrowth factors described above. Differentiation and modulation ofdifferentiation can be carried out in accordance with known techniques,e.g., as described in U.S. Pat. No. 6,589,728, or U.S. PatentApplication Publication Nos.: 2006006018 (endogenous repair factorproduction promoters); 20060013804 (modulation of stem celldifferentiation by modulation of caspase-3 activity); 20050266553(methods of regulating differentiation in stem cells); 20050227353(methods of inducing differentiation of stem cells); 20050202428(pluripotent stem cells); 20050153941 (cell differentiation inhibitingagent, cell culture method using the same, culture medium, and culturedcell line); 20050131212 (neural regeneration peptides and methods fortheir use in treatment of brain damage); 20040241856 (methods andcompositions for modulating stem cells); 20040214319 (methods ofregulating differentiation in stem cells); 20040161412 (cell-based VEGFdelivery); 20040115810 (stem cell differentiation-inducing promoter);20040053869 (stem cell differentiation); or variations of the above orbelow that will be apparent to those skilled in the art.

“Subjects” are generally human subjects and include, but are not limitedto, “patients.” The subjects may be male or female and may be of anyrace or ethnicity, including, but not limited to, Caucasian,African-American, African, Asian, Hispanic, Indian, etc. The subjectsmay be of any age, including newborn, neonate, infant, child,adolescent, adult and geriatric subjects.

Subjects may also include animal subjects, particularly vertebratesubjects, e.g., mammalian subject such as canines, felines, bovines,caprines, equines, ovines, porcines, rodents (e.g., rats and mice),lagomorphs, non-human primates, etc., or fish or avian subjects, for,e.g., veterinary medicine and/or research or laboratory purposes.

“Treat” refers to any type of treatment that imparts a benefit to asubject, e.g., a patient afflicted with a trauma or disease. Treatingincludes actions taken and actions refrained from being taken for thepurpose of improving the condition of the patient (e.g., the promotionof healing and/or formation of tissues on a patient in need thereof, therelief of one or more symptoms, etc.). In some embodiments, treatingincludes reconstructing skin tissue (e.g., where such tissue has beendamaged or lost by injury or disease) by directly printing cells and/ortissues onto a subject in need thereof.

The present invention provides for the printing of tissues by theappropriate combination of cell and support material, or two or three ormore different cell types typically found in a common tissue (e.g., skintissue). Cells, support compounds, and growth factors may be printedfrom separate nozzles or through the same nozzle in a commoncomposition, depending upon the particular tissue (or tissue substitute)being formed. Printing may be simultaneous, sequential, or anycombination thereof. Some of the ingredients may be printed in the formof a first pattern (e.g., an erodable or degradable support material),and some of the ingredients may be printed in the form of a secondpattern (e.g., cells in a pattern different from the support, or twodifferent cell types in a different pattern). Again, the particularcombination and manner of printing will depend upon the particulartissue. Materials to be printed for specific tissues or tissuesubstitutes are described further below.

Skin.

In representative embodiments, to produce epidermal-like skin tissue,the following are printed:

-   -   (a) at least one cell type, and preferably at least two or in        some embodiments three or four different epidermal cell types        (e.g., keratinocytes, melanocytes, Merkel cells, Langerhan        cells, etc., and any combination thereof); and/or    -   (b) at least one support compound such as described above (e.g.,        collagen, elastin, keratin, etc., and any combination thereof);        and/or    -   (c) at least one growth factor as described above (e.g., basic        fibroblast growth factor (bFGF), Insulin-Like Growth Factor 1,        epidermal growth factor (EGF), etc., and any combination        thereof);

In some embodiments the epidermal cells, support compound and/or growthfactors printed as described above (which form an “epidermal” typelayer) are printed on, or on top of, a previously formed (e.g., printedor ink-jet printed) “dermal” type layer, the previously printed dermallayer layers comprising: (a) one, two, three or four different dermalcells (fibroblasts, adipocytes, mast cells, and/or macrophages), (b) atleast one support compound as described above; and/or (c) at least onegrowth factor as described above.

Skin tissue produced by the process of the present invention is usefulfor treatment of, for example, burns, and other wounds such asincisions, lacerations, and crush injuries (e.g., postsurgical wounds,and posttraumatic wounds, venous leg ulcers, diabetic foot ulcers,etc.).

Bone.

In particular embodiments, to produce bone tissues, the following areprinted:

-   -   (a) at least one bone cell type, and preferably at least two or        three different bone cell types (e.g., osteoblasts, osteoclasts,        osteocytes, and any combination thereof, but in some embodiments        at least osteoblasts and osteoclasts, and in some embodiments        all three); and/or    -   (b) at least one support compound such as described above (e.g.,        collagen, hydroxyapatites, calicite, silica, ceramic,        proteoglycans, glycoproteins, etc., and any combination        thereof); and/or    -   (c) at least one growth factor (e.g., bone morphogenetic        protein, transforming growth factor, fibroblast growth factors,        platelet-derived growth factors, insulin-like growth factors,        etc., and any combination thereof).

Bone tissues produced by the processes described herein are useful for,among other things, implantation into a subject to treat bone fracturesor defects, and/or promote bone healing.

Nerve.

In representative embodiments, to produce nerve tissue, the followingare printed:

-   -   (a) at least one, two or three cells types, and preferably (i)        peripheral nerve cells and/or (ii) at least one glial cell type,        and (iii) any combination thereof (e.g., a combination of at        least one nerve cell and at least one glial cell); and/or    -   (b) at least one support compound such as described above;        (e.g., laminin, collagen type IV, fibronectin, etc., and any        combination thereof); and/or    -   (c) at least one growth factor (e.g., NGF, brain-derived        neurotrophic factor, insulin-like growth factor-I, fibroblast        growth factor, etc., or any combination thereof); and any        combination of the foregoing.

Nerve tissue produced by the processes described herein is useful, amongother things, to treat nerve injury or degenerative diseases affectingthe peripheral nervous system.

Muscle.

In representative embodiments, to produce muscle tissue, the followingare printed:

-   -   (a) at least one muscle cell type; and/or    -   (b) at least one support compound such as described above;        (e.g., laminin, collagen type IV, fibronectin, etc., and any        combination thereof); and/or    -   (c) at least one growth factor (e.g., vascular endothelial        growth factor, insulin-like growth factors (IGFs), etc., or any        combination thereof); and any combination of the foregoing.

Muscle tissue produced by the processes described herein is useful,among other things, to treat smooth muscle, skeletal muscle or cardiacmuscle injury or diseases affecting these tissues.

Vascular Tissue.

In representative embodiments, to produce vascular tissue, the followingare printed:

-   -   (a) at least one vascular cell type, and preferably at least two        or three different vascular cell types (e.g., endothelial cells,        smooth muscle cells, fibroblasts, and any combination thereof,        but in some embodiments at least endothelial cells, smooth        muscle cells, and in some embodiments all three); and/or    -   (b) at least one support compound such as described above;        (e.g., laminin, collagen type IV, fibronectin, etc., and any        combination thereof); and/or    -   (c) at least one growth factor (e.g., vascular endothelial        growth factor, insulin-like growth factors (IGFs), etc., or any        combination thereof); and any combination of the foregoing.

Vascular tissue produced by the processes described herein is useful,among other things, to form vascular networks and/or treat injury ordiseases affecting these tissues.

In some embodiments, the tissue is created “in sequence” layer-by-layer,with a printed layer (A), then a printed layer (B), and so on as neededin series, such as layers:

A B C D . . .

Each layer may comprise cells, support compounds, growth factors,combinations thereof, etc., as desired to construct the tissue as neededor desired.

In some embodiments, cells may be printed in a first layer, followed bya second layer of support materials such as a gel, optionally followedby a third layer of cells. For example, a multiple layered skin tissuemay be printed as a layer comprising fibroblasts, followed by a layercomprising a gel (e.g., comprising fibrin, fibrinogen, collagen, etc.),followed by a layer comprising keratinocytes. Additional layers may alsobe provided as desired. Thrombin may also be printed with or onto one ormore layers, if desired.

In some embodiments, skin cells and/or layers thereof can be printed(e.g., fibroblasts, keratinocytes, melanocytes, etc.), and additionalskin cells printed thereon or therewith in discrete units and/orpatterns. For example, papilla cells, which form hair follicles, in someembodiments may be printed at specific locations and/or densities (hairshaft thickness and length being at least in part determined by dermalpapilla cell number and volume, with hairs becoming longer withincreasing cell number), which can generate hair more closelyapproximating that from the native skin tissue for different bodylocations, age groups, genders, etc. Outer root cells may also beprinted with the dermal papilla cells, if desired. Melanocytes may alsobe printed at specific locations and/or densities as desired, forexample, to better match adjoining skin (e.g., with freckles).

In some embodiments, use of these cell printing patterns may also aid inregeneration of old wounds that already have scars, replacing the scarswith more natural-looking skin.

D. Methods of Data Processing, Computer Programs and Systems.

In some embodiments, data obtained from the optical detector is piecedtogether to form a model of the bodily surface of interest (e.g., awound surface). The bodily surface may be of any body portion, includinga hand, foot, arm, leg, torso, chest, abdomen, back, head, face,portions thereof and/or combinations thereof, etc., including a woundarea on the same.

In some embodiments, the surface is then transformed into a mold of thesurface. A “mold” as used herein is a three-dimensional representationof the bodily surface of interest, which in some embodiments may bedisplayed visually, and which may also include, in addition tothree-dimensional map coordinates, information such as color and/orinfrared radiation intensity, among others. The mold according to someembodiments is interpreted into a “negative” mold, in which the layersare reversed, and may be further split into layers on the Z axis todetermine which layers correspond to the natural tissue layers (e.g.,dermis and epidermis layers of skin tissue), as necessary to guide theassociated printer.

In some embodiments, each layer is overlaid with a series of lines(i.e., the lines are added to the layer representation or mold) thatcover at least a portion of the area detected. These lines may then beused (e.g., by control software) to determine a path for the printer.The printer may print along the series of lines for each layer, thusforming a tissue in a layer-by-layer fashion.

In some embodiments, the user can define a series of bitmap images whereeach non-black pixel corresponds to a cell drop, and the color of thepixel corresponds to the cell type to print. This allows the user tocreate complex structures using various cell types and/or in variousconfigurations.

System and/or component operation according to some embodiments may beimplemented by computer program instructions. These computer programinstructions may be provided to a processor of a general purposecomputer, special purpose computer, and/or other programmable dataprocessing apparatus to produce a machine, such that the instructions,which execute via the processor of the computer and/or otherprogrammable data processing apparatus, create means for implementingthe desired operations.

These computer program instructions may also be stored in acomputer-readable memory that can direct a computer or otherprogrammable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including instructions whichimplement the operations.

The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer-implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing the operations.

Accordingly, the present invention may be embodied in hardware and/or insoftware (including firmware, resident software, micro-code, etc.).Furthermore, embodiments of the present invention may take the form of acomputer program product on a computer-usable and/or computer-readablestorage medium having computer-usable or computer-readable program codeembodied in the medium for use by or in connection with an instructionexecution system. As used herein, a computer-usable or computer-readablemedium may be any medium that can contain or store the program for useby or in connection with the instruction execution system, apparatus, ordevice.

The computer-usable and/or computer-readable medium may be, for examplebut not limited to, an electronic, magnetic, optical, electromagnetic,infrared, or semiconductor system, apparatus or device. More specificexamples (a non-exhaustive list) of the computer-readable medium wouldinclude the following: a portable computer diskette, a random accessmemory (RAM), a read-only memory (ROM), an erasable programmableread-only memory (EPROM or Flash memory), and a portable compact discread-only memory (CD-ROM).

In some embodiments, software employs a three-tiered architecturedesign. The three-tiered architecture handles three areas ofcommunication: between the user and the software; among the softwarecomponents; and between the software and the file system. This designstructure may allow individual components to be quickly and easilyaltered as necessary without affecting the other components.

In some embodiments, data gathered from the optical detector arerepresented as an object (e.g., a skin object). Each object may compriseone or more layers of the tissue, and each layer may be broken into agrid that encapsulates the individual lines from detector. Thisrepresentation may significantly reduce the time necessary to deliverskin by allowing the delivery system to print points from one piece ofthe grid while the computer analyzes the next piece. In someembodiments, points from the object are passed in a generic format tothe delivery system where they are parsed for printing in a manner thatincorporates the locations of the different cell types in the printhead.This implementation may allow the user to quickly define enhancements tothe scanned data and include other components of the tissue (e.g., skincomponents such as melanocytes and hair follicles for skin tissue).

The skin delivery system may be controlled by software employing athree-tiered architecture design. In some embodiments, the architecturemay be based on the Microsoft .NET Framework 2.0 (Microsoft Corp.,Redmond, Wash.). Some embodiments provide that the software may bewritten in any one or combination of programming languages, includingobject-oriented programming languages such as, for example, C++, amongothers. The three-tiered architecture may be configured to handle threeareas of communications: between the user and the software; among thesoftware components; and between the software and the file system. Thisdesign structure may allow every component to be quickly and easilyaltered as necessary without affecting the other components.Furthermore, because the software is not based on a proprietary systemsuch as, for example, MATLAB®, it can be deployed to any computercapable of running the .NET Framework 2.0.

The present invention is explained in greater detail in the followingnon-limiting examples.

Example 1

This example describes the design and use of a novel delivery system forin situ bioprinting of the skin. The cartridge based system presentedhere can be easily transported from patient to patient and can rapidlyprint skin constructs that mimic normal skin. The device allows forrapid on-site management of burn wounds, integrating a method todetermine the size, shape, and depth of a wound with controlled deliveryof skin cells to the target wound site in situ. This integration allowsthe system to effectively manage the treatment of large injuries whilereconstructing the normal skin structure. A proof-of-concept experimentusing in situ delivery of fibroblasts and keratinocytes to a mouse modelof large skin wounds described below demonstrates its efficacy.

Materials and Methods

The following criteria were established to guide the hardware design forthis exemplary system:

1. The system should be portable and capable of being quicklytransported to the wounded personnel and it should be easily convertedfor use in different patients with different needs. For example, thesystem should be capable of fitting through hospital doors and hallways,and it should be constructed from lightweight materials for easytransport.2. The system should be capable of easy sterilization.3. The system should be capable of tailoring cell therapy to a patient'sspecific needs.4. The system should allow for a wide range of body types.5. The system should be capable of repeated use.6. Maintenance of the system should be relatively easy and inexpensive.

We have accomplished these goals by using a cartridge based deliverysystem with a laser scanning system, both mounted on a portable XYZplotting system (FIG. 1). The cartridge system is similar to that usedin traditional inkjet printing such that each cell type is loaded intoan individual cartridge in the same way different color inks would becontained in different cartridges. However, standard inkjet printingconnects one printhead to one cartridge, while the skin delivery systemallows each cartridge to connect to multiple printheads. This type ofconfiguration allows arbitrary printhead configurations that can conformto each patient's specific needs. It also increases the throughput ofthe system and provides a rapid method of sterilization by attaching acartridge of cleaning fluid to the printheads.

The printheads in the novel device use pressure-based nozzles instead ofthe thermal or piezoelectric microfluidic delivery devices used intraditional inkjet printers. A pressure-based delivery system allows theprinter to remain a safe distance above the patient to accommodate avariety of body types.

Tailoring cell therapy to individual patients requires length, width,and depth information about the wound. This system incorporates athree-dimensional laser scanner (NextEngine Inc., Santa Monica, Calif.)mounted above the patient. This scanner can be moved to variouslocations on the body, and information about the wound is gathered withan accuracy of approximately 127 μM.

The cartridges, printheads, and scanner are mounted on the Z axis of abelt-driven plotting system (FIG. 1) capable of 100 μm movements. The Yaxis moves the X axis which in turn moves the Z axis. Since the X and Yaxes comprise the majority of the system's weight this configurationallows those axes to remain stationary while permitting the system toaccommodate a wide range of body types.

The plotting system is mounted on a mobile frame. Patients with massiveburn injuries are difficult to transport and some current treatments,including INTEGRA® and autologous keratinocyte culture, require multipleprocedures. Multiple procedures require the fragile patient to be movedbetween the bed and the operating room for a number of times. This frameis designed to alleviate transport concerns by allowing the system toquickly move to patient beds and operating rooms.

Software

The skin delivery system is controlled by software employing athree-tiered architecture design based on the Microsoft .NET Framework2.0 (Microsoft Corp., Redmond, Wash.) and was written in C++. Thethree-tiered architecture handles three areas of communication: betweenthe user and the software, among the software components, and betweenthe software and the file system. This design structure allows everycomponent to be quickly and easily altered as necessary withoutaffecting the other components. Furthermore, because the software is notbased on a proprietary system such as MATLAB®, it can be deployed to anycomputer capable of running the .NET Framework 2.0.

The data obtained from the laser scanner is pieced together to form amodel of the wound surface. This surface is transformed into a negativemold of the wound which is split into layers on the Z axis to determinewhich layers correspond to the dermis and epidermis. Each layer isoverlaid with a series of lines that cover the entire wound area. Theselines are used by the control software to determine a path for theprinter.

Each of the smaller components of the software follows a design patternsimilar to the overall architecture. Data gathered from the laserscanner are represented as a skin object. Each skin object consists ofskin layers, and each layer is broken into a grid that encapsulates theindividual lines from the scanner. This representation significantlyreduces the time necessary to delivery skin by allowing the deliverysystem to print points from one piece of the grid while the computeranalyzes the next piece. Points from the skin object are passed in ageneric format to the delivery system where they are parsed for printingin a manner that incorporates the locations of the different cell typesin the printhead. This implementation allows the user to quickly defineenhancements to the scanned data and include other important skincomponents such as melanocytes and hair follicles.

Animal Model and Testing

All animal procedures were performed according to the protocols approvedby the Wake Forest University Health Sciences Animal Care and UseCommittee. The skin delivery system prototype was evaluated by creatinga 3×2.5 cm (L×W) full-thickness skin defect on the dorsa of six femaleoutbred athymic nude (Nu/nu) mice (Charles River Laboratories, Raleigh,N.C.). This defect represents approximately a 50% TBSA wound. Three micewere untreated and the others were treated by cell printing. Humanfibroblasts were obtained from human foreskin and cultured in highglucose Dulbecco's modified Eagle's medium (Gibco-BRL, Grand Island,N.Y.) with 5% fetal bovine serum and 1% antibiotics. Human keratinocyteswere obtained from ScienCell (Carlsbad, Calif.) and cultured inkeratinocyte serum-free media (Gibco-BRL) with 1% antibiotics. Whensufficient cell numbers were reached, cells were trypsinized for 5 minand suspended in a mixture of 25 mg/mL fibrinogen and 1.1 mg/mL type Icollagen in phosphate-buffered saline. One layer of fibroblasts (passage6) was printed at 250,000 cells cm⁻² followed by an equal amount of 20IU/mL thrombin. The thrombin was allowed to react for 15 minutes beforea layer of keratinocytes (passage 5) was printed at 500,000 cells cm⁻²,again followed by thrombin. Each wound received antibiotic cream and wascovered with sterile cotton gauze wrapped in surgical tape to preventremoval by the mouse. Wound coverings were changed at 1 week and removedat 2 weeks post-surgery. Fibroblasts and keratinocytes given to onemouse were prelabeled with the fluorescent dyes PKH 26 (red) and PKH67(green) (Sigma-Aldrich, St. Louis, Colo.), and this mouse along with oneuntreated mouse were sacrificed at 1 week for retrieval of the woundarea to determine if the labeled cells were present in the construct.The wound size for the experiment with labeled cells was 1.5×2 cm (L×W)for proof of concept. For the other two mice, each 3×2.5 cm wound wasevaluated every week for 3 weeks to determine the size of the wound andthe extent of scarring. At 3 weeks the scar tissue was removed forhistological evaluation.

Results

The skin delivery system is capable of printing skin cells directly ontoa full-thickness defect on nude mice. Skin constructs printed withfluorescent prelabeled cells and retrieved after 1 week showed thepresence of labeled cells within the wound bed (data not shown). Thesecells appeared to participate in the healing process, as near-completeclosure of the wound occurred at 2 weeks, and complete wound closureoccurred at 3 weeks (data not shown). H&E staining of the skinconstructs at 3 weeks demonstrated structural similarity to normal skin,with organization of the keratinocytes into epidermal strata and thefibroblasts into dermis (data not shown). The untreated mouse showedwound healing in the same timeframe but did not demonstrate completewound closure as seen in the printed mice. In addition, the center ofthe untreated wound shows inflammation and scabbing at 3 weeks whereasthe covered wound shows cellular integration into the surrounding skin.Results are shown in FIG. 9.

Discussion

Our skin delivery system allows rapid production of patient-specificwound coverage while simultaneously obviating the need for specializedmanufacturing facilities and cell culture materials at burn carecenters. Furthermore, the delivery system fulfills many of the criteriafor an ideal skin substitute as demonstrated by previous uses ofallogeneic skin cells for burn coverage. Cells printed using thedelivery system adhere intimately to the wound bed, provide anon-antigenic microbial barrier, participate in normal host repairmechanisms, maintain elasticity and long-term durability, displaylong-term mechanical and cosmetic function comparable to split-thicknessautografts, require a single operation, are inexpensive, and haveminimal storage requirements. The only ideal skin substitutecharacteristic that is not fulfilled by this system is the requirementof indefinite storage, which is virtually impossible to achieve with anyliving skin substitute. However, the cartridge system allows packing andshipping of allogeneic cells to burn centers, which in turn allowstreatment of the wound as soon as the patient is stable and the woundhas undergone debridement. In contrast, a typical autologous graftrequires 2-5 weeks to grow in culture.

We demonstrated proof-of-concept by printing a two-layer skin constructconsisting of fibroblasts and keratinocytes directly into afull-thickness skin defect on a nude mouse. This printing was performedby delivering specific cells to specific target areas.

We embedded the cells in a matrix of fibrinogen and type I collagen fortwo reasons. First, the formation of fibrin from the reaction offibrinogen and thrombin provides a strong gel that allows the cells tomaintain their position on the mouse even if the mouse moves. Second,fibrin and type I collagen have already been used to create skinconstructs. After printing, prelabeled fibroblasts and keratinocyteswere visible in the construct 1 week post-printing, indicating that thecells remained in the wound area. Evaluation of the wound area over 3weeks showed rapid closure of the wound in the treated mouse as comparedto the untreated mouse. The remaining open wound in the printed group at2 weeks post-surgery was open only at the center of the wound in thearea of greatest body curvature. This could be due to the possibilitythat the printed cell droplets rolled off the curvature. One way tocorrect for this may be by replacing the thrombin delivery system with anebulizer to rapidly create fibrin.

H&E staining of the printed constructs showed organization of the cellsinto a structure similar to normal skin. The epidermis was the samethickness in both the printed construct and normal tissue. There was ademarcation at the dermal-epidermal junction and the dermis in theprinted construct appeared to be similar in composition to the normaltissue. This shows the ability of the skin delivery system to printtissue that mimics the normal skin structure.

While this study only examined the use of fibroblasts and keratinocytesin skin printing, the design of the system allows for precise deliveryof additional cell types. These include, but are not limited to,follicular cells, melanocytes, and endothelial cells. Including theseadditional cell types could further mimic the normal skin structure andprovide functional and cosmetic improvements over current treatmenttechniques, especially with regard to pigmentation and vascularization.The print cartridges can also be designed to include factors aimed atimproving the function of the skin constructs. These include scarlesshealing reagents, growth factors, and protease inhibitors to maintainthe longevity of other reagents. If a cell type or reagent can bepackaged into a cartridge, our system can rapidly deliver that cell orreagent to a specific location on the patient. This property makes oursystem superior to most current burn treatments because it eliminatesthe need for culturing cells and reagents in a graft construct prior topatient transplantation.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A system for processing data of a bodilysurface obtained from a three-dimensional optical detector to provide apath to a printer operatively associated to said optical detector, saidsystem comprising: means for interpreting data from said opticaldetector to form a model of the bodily surface; means for transformingsaid model into a negative mold of the bodily surface, which mold issplit into a plurality of Z-axis layers; and means for overlaying eachof said layers of the negative mold with a series of lines whichrepresents coverage of the bodily surface, wherein said lines provide apath for control software to use to control the printer to deliver cellsand/or compositions to said bodily surface in situ based upon saidbodily surface data, wherein the bodily surface data is wound surfacedata, and wherein said data is updated in real-time during delivery ofthe cells and/or compositions by said printer.
 2. The system of claim 1,further comprising means for obtaining the data.
 3. The system of claim1, wherein the data is skin wound surface data, and wherein the Z-axislayers correspond to tissue layers comprising dermis and/or epidermislayers of skin tissue.
 4. The system of claim 1, wherein the data arerepresented as an object including the Z axis layers, and wherein eachof the Z axis layers are represented by a grid which comprises theseries of lines.
 5. The system of claim 4, further comprising means forparsing the object in a manner that incorporates locations of differentcells and/or compositions to be printed by the printer.
 6. The system ofclaim 1, wherein said Z-axis layers correspond to one or more tissuelayers.
 7. A computer program product for processing data of a bodilysurface obtained from a three-dimensional optical detector to provide apath to a printer operatively associated to said optical detector, thecomputer program product comprising a non-transitory computer readablemedium having computer readable program code embodied therein, thecomputer readable program code comprising: computer readable programcode which interprets the data of the bodily surface from said opticaldetector to form a model of the bodily surface; computer readableprogram code which transforms said model into a negative mold of thebodily surface, which mold is split into a plurality of Z-axis layers;and computer readable program code which overlays each of said Z-axislayers of the negative mold with a series of lines which representscoverage of the bodily surface, wherein said lines provide a path forcontrol software to use to control the printer to deliver cells and/orcompositions to said bodily surface in situ based upon said data,wherein the bodily surface data is wound surface data, wherein said datais updated in real-time during delivery of the cells and/or compositionsby said printer, wherein the data are represented as an object includingthe layers, wherein each of the layers are represented by a grid whichcomprises the series of lines, and wherein the computer readable programcode is configured to direct the printer to deliver cells to a firstlocation of the bodily surface associated with a first piece of the gridrepresenting the object while analyzing a second piece of the grid. 8.The computer program product of claim 7, further comprising computerreadable program code which parses the object in a manner thatincorporates locations of different cells and/or compositions to beprinted by the printer.
 9. The computer program product of claim 7,wherein said Z-axis layers correspond to one or more tissue layers. 10.The computer program product of claim 9, wherein said bodily surface isa skin wound surface, and wherein the tissue layers comprise dermisand/or layers of skin tissue epidermis layers of skin tissue.